Session 10: The Immune Response in Health and Disease

Transcript of Part 3: The Troublesome Tubercle in Tuberculosis

00:00:07.21	My name is Lalita Ramakrishnan.
00:00:09.16	I'm a professor of Immunology and Infectious diseases at the University of Cambridge.
00:00:14.08	And, in this lecture, I'm going to continue to tell you a little bit more about tuberculosis,
00:00:20.16	focusing on a structure called the tubercle.
00:00:24.13	Now, just to recapitulate the lifestyle or the life cycle of Mycobacterium tuberculosis,
00:00:33.02	which is the causative agent of tuberculosis, the bacterium that causes it, the bacteria
00:00:39.01	are exhaled by... in coughs by people who are infected, get inhaled by individuals near
00:00:48.22	them, and then they enter cells that are called macrophages.
00:00:52.18	But what happens next is that they... they induce these macrophages to form structures
00:01:01.16	called granulomas, and these granulomas can become quite elaborate.
00:01:09.14	At first, they're comprised just of macrophages, but then many other immune cells come in and
00:01:15.13	they can form of a fairly complex structure.
00:01:19.21	And another thing to note is that the macrophages within this granuloma undergo a specialized
00:01:27.15	differentiation called epithelioid transformation, where they form these finger-like, interdigitated
00:01:34.03	projections between each other to form a very compact, organized structure.
00:01:42.23	Now, this structure... pathologists call these structures granulomas, and granulomas were...
00:01:51.22	are... are associated with many, many, many diseases, both infectious and non-infectious.
00:01:59.19	And they can often be quite pathological.
00:02:02.03	But granulomas... but... but the single biggest cause of granulomas is tuberculosis.
00:02:09.13	And, in fact, granulomas were first discovered in the context of tuberculosis in 1679, some
00:02:18.13	200 years before the bacterium Mycobacterium tuberculosis was... was discovered.
00:02:25.09	And, in fact, the microbe... the... the structure used to be called a tubercle, and obviously
00:02:31.23	that... both the bacterium and the disease are named for the granuloma.
00:02:35.16	So... but it turns out that granulomas are very primitive structures; they've probably
00:02:43.03	evolved... there... they're present even in lower vertebrates in a... in a more primitive
00:02:47.11	form, and they probably evolved to wall off foreign bodies -- you could imagine a thorn
00:02:54.02	being... being circumscribed by that... by such a structure until it's...
00:02:59.03	it's sort of dissolved.
00:03:01.00	But these so-called foreign body... body granulomas have a very low turnover of macrophages; the
00:03:07.14	macrophages just come and sit there and it's not a particularly inflammatory structure,
00:03:11.14	or so it's thought.
00:03:13.19	In contrast, the types of granulomas that form with tuberculosis, and pretty much any
00:03:18.02	medically significant granuloma, tend to be high turnover granulomas where there's a rapid
00:03:25.10	death of macrophages and a repopulation by new ones,
00:03:28.22	and they can be very inflammatory structures.
00:03:31.22	There are many traditional animal models that are used to study TB.
00:03:36.11	The oldest ones are the ones... the rabbit and the guinea pig, which were used by
00:03:42.19	Villemin and Koch, respectively, at the time that they discovered TB.
00:03:48.11	They used these animals to... to pass the bacterium from one animal to another to show
00:03:54.19	that it was associated with... with TB.
00:03:58.18	The most commonly used model now is the... is the mouse.
00:04:01.21	And this makes a lot of sense because mice have a wonderful array of
00:04:08.03	immunological and genetic tools.
00:04:10.06	One issue is that mice don't... that most mice... mouse strains don't develop the...
00:04:18.07	the nice, tight granulomas that are associated with human disease, but there are some recently
00:04:25.06	identified mouse strains that do, and so those could actually be quite good.
00:04:33.07	Another more recently used model is the non-human primate, and this is a good model because
00:04:40.04	it really recapitulates human disease.
00:04:43.03	But the problem is, of course, they're expensive, there are ethical considerations, and obviously
00:04:48.06	cannot be used widely.
00:04:50.19	So, in... on this backdrop, my story and my engagement with TB and the granuloma came
00:05:00.24	when, as a postdoctoral fellow at UCSF -- I was a clinical infectious disease fellow and
00:05:07.08	had to do a postdoctoral fellowship -- I approached Stanley Falkow at Stanford to... to go to
00:05:14.07	his lab and study TB, which he didn't study at the time,
00:05:17.14	but he studied many other bacterial pathogens.
00:05:20.22	And Stanley said to me, forget it, I don't have the specialized containment facilities
00:05:27.00	you need to study TB, which is a human aerosol pathogen and, besides, he said, TB grows so
00:05:33.05	slowly I'll be dead before you get your first result.
00:05:36.18	And that was in 1991, and I'm happy to say he's still alive, and... and...
00:05:42.17	and quite well.
00:05:45.13	And... so... what he told me... he gave me an insight and he said, look, there are other
00:05:53.19	strains of mycobacteria, there are other species of mycobacteria that are pathogenic in other
00:05:59.01	animals, that are natural pathogens of other animals.
00:06:02.20	And he said, I...
00:06:03.20	I'm pretty sure there are these ones of... of marine life of fish, because I've seen
00:06:09.19	people get them who were fishermen.
00:06:13.19	He had worked at Brown and knew that Portuguese fishermen got these... this disease... mycobacterial
00:06:20.15	disease on their digits and on their soft tissues.
00:06:24.22	And so I went off to the UCSF library and looked at this very classic manual.
00:06:31.13	It's called Bergey's Manual of Systemic Bacteriology, and I... and I found what he was talking about.
00:06:37.16	He was talking about... he was probably talking about a bacterium called Mycobacterium marinum
00:06:43.13	that was thought to be a close relative of human TB, of the human TB bacterium, and it
00:06:50.01	gave fish TB.
00:06:52.24	It turns out that it also infects humans, and this has been known since the 50s, and
00:07:00.02	I can personally attest to it.
00:07:02.18	And it gives humans disease on their extremities, as Stanley already knew, and... and many clinicians,
00:07:08.23	particularly dermatologists and infectious disease clinicians, already know this.
00:07:13.12	But if you look inside that lesion, you'll see a classic granuloma and actually in many
00:07:18.20	cases it can be indistinguishable from the granulomas caused by the human TB bacterium.
00:07:27.03	But, of course, we know that this bacterium also infects fish, and it was first identified
00:07:34.03	to do so in the Philadelphia Aquarium, where in 1926 fish were dying of some mysterious
00:07:42.20	wasting disease, very similar to human TB.
00:07:46.12	And when they tried to culture these fish to see what bacterium they had, why were they
00:07:52.23	dying?, they couldn't culture anything, but when they looked at the fish by histology
00:07:57.24	they could see these classic red snapper bacteria that looked very much like TB.
00:08:04.05	And then Aronson had the bright idea to culture the... to try to do the cultures at a low
00:08:09.21	temperature that was commensurate with... with the low body temperature of the fish,
00:08:14.02	and then he was able to cultivate, he was able to culture Mycobacterium marinum.
00:08:19.08	And, since then, we've had Mycobacterium marinum sequenced at the Sanger Center, and it turns
00:08:26.22	out to be the closest genetic relative of the human TB bacterium, so I guess we also
00:08:32.16	got quite lucky.
00:08:34.00	And it turns out that Mycobacterium marinum also infects zebrafish.
00:08:39.14	Zebrafish are a pet develop... a pet organism of developmental biologists and are a natural
00:08:46.09	host to Mycobacterium marinum.
00:08:48.20	So, I've got for you, here, down below in... in the bottom panel, the... a human TB granuloma
00:08:56.14	stained by hematoxylin and eosin, which will only stain the host cells but not the bacteria,
00:09:01.20	and what you can see is that you've got a nice organized structure, which is cellular,
00:09:07.23	as evidenced by blue nuclei on the edges, but in the center where that arrowhead is,
00:09:14.12	you'll see that the structures become acellular, because it's undergone necrosis, just as we
00:09:21.20	know that human TB granulomas do.
00:09:24.03	But here's, now... let's take a look, now, at the... at a zebrafish granuloma, and in
00:09:30.03	this granuloma you can see... which... which I've stained, here, with a stain that also
00:09:36.22	stains the bacteria, you can see that it looks very similar and it's a nice cellular organized
00:09:43.13	structure and you can see that there are a few bacteria within macrophages, but where
00:09:48.12	the macrophages have necrosed there are tons of bacteria,
00:09:51.12	which is exactly what you would expect.
00:09:55.11	But the great feature of the zebrafish that makes them so enticing to developmental biologists
00:10:01.11	is that they have a prolonged larval phase when they're transparent.
00:10:06.09	And so you can actually watch things happen and people watch developmental processes happen,
00:10:12.24	but... so we asked, well, can we put in bacteria and watch infection happen?
00:10:18.10	And so, they have a cavity that's called the hindbrain ventricle, which is the equivalent
00:10:22.24	of something in our brain, close to our brain called the fourth ventricle, and so we put
00:10:27.09	in some bacteria there -- I've shown it to you with an arrowhead, there -- and what we
00:10:32.03	saw very quickly was that macrophages came, and you'll see the macrophages sort of chasing
00:10:40.14	after the bacterium like a cat after a mouse, and eventually you'll see this mac... mac...
00:10:45.17	macrophage gets it.
00:10:47.12	And there you've got an infected macrophage.
00:10:51.20	You can now follow these infected macrophages out of the cavity -- this is a few days later
00:10:57.12	-- and you can see that it's just moseying along.
00:11:02.04	The bacteria have grown in the macrophage and -- because it's a permissive macrophage
00:11:08.19	for the... for the... for the bacterium -- and there it is.
00:11:13.20	But what was really exciting to us was that you could see, within a few days,
00:11:19.02	a granuloma form.
00:11:20.22	And here you can see that we've got a granuloma that's already formed and what you're going
00:11:25.08	to see, where that white... white arrow is, a new uninfected macrophage is going to come
00:11:32.00	and then it's going to enter the structure.
00:11:34.20	So, watch this.
00:11:37.13	See?
00:11:39.11	There it comes, and it's going to squeeze its way in between and get in there.
00:11:50.21	So, the granuloma is a highly chemotactic structure
00:11:54.21	that is recruiting new macrophages to come to it.
00:11:59.19	And then we could show all this by... by engineering fish that were transgenic, so that they had
00:12:07.06	green florescent macrophages and red fluorescent neutrophils, which is another cell type that
00:12:12.11	is somewhat involved in granulomas, but not as much as macrophages.
00:12:16.17	And what you can... and now we've infected the fish with blue fluorescent bacteria and
00:12:20.10	you can see that the... that we've got a nice tight bona fide epithelioid granuloma with
00:12:28.00	infected macrophages.
00:12:29.13	So, this was good.
00:12:31.10	As we were developing this model, my colleague and friend, David Sherman,
00:12:37.11	made a suggestion to us.
00:12:40.16	He... so, it's... people have been searching for virulence determinants in mycobacterium,
00:12:50.03	and one exciting discovery was that a specialized secretion system called the ESX-1 or RD1 locus,
00:12:58.18	which I've shown in white in that top panel, the white genes in the top panel,
00:13:03.24	were involved in virulence.
00:13:05.16	And this was very exciting because these... it turns out that this was the locus that
00:13:10.09	was missing in the attenuated vaccine strain, BCG, that was made by serial passage in...
00:13:17.14	in the 1920s, and now we finally knew the molecular basis of its attenuation.
00:13:24.19	So, Mycobacterium marinum, not surprisingly, has a locus that looks virtually identical.
00:13:31.19	And David kept telling me, look, make a mutation in this and let's see how it really works,
00:13:38.10	because everyone knows it's attenuated, but a lot of these animal models are black boxes
00:13:42.24	because you only get to see the end result, and he could see that we would be able to
00:13:47.01	get some insights about the actual sequence of what was got... what was different.
00:13:52.04	And, when I was a bit slow to do this, he actually had someone in his lab make the mutation...
00:13:59.04	the mutant for me, and he gave it to us and he said, take this.
00:14:02.02	So, at this point, we were sort of shamed into doing this quickly, and we was
00:14:07.03	Hannah Volkman, who had joined my lab at... as a graduate student, and Hannah showed very quickly
00:14:12.08	that, yes, if you put this mutant into zebrafish larva, it was attenuated.
00:14:17.16	The animals didn't die and if you looked at the bacterial counts you could see that the
00:14:23.03	bacteria didn't grow as well.
00:14:24.03	So, this was good because it showed us that it was behaving just like you would expect.
00:14:28.12	But, here came the surprise.
00:14:30.21	And, at this point... by this point, Hannah had recruited some of her colleagues -- Dana
00:14:36.12	Beery, on the left, who was a technician in the lab, and Hilary Clay, a graduate student
00:14:41.01	who was Hannah's very good friend -- and she got them to join in this... in this quest
00:14:46.01	to see what was going on.
00:14:48.15	And what they found was something quite interesting.
00:14:52.13	Because, if you look at the fish on top, they are infected with wild-type bacterium, and,
00:14:58.19	if you look at the close-up on the top right,
00:15:01.17	you can see that a nice big granuloma has formed.
00:15:04.22	But if you look at the mutant, what you can see is that, even if you inject many, many
00:15:09.22	more bacteria, just to compensate, so that you get more... as many bacteria as with the
00:15:16.10	wild-type, the macrophages pack up with the bacteria, as you see on that bottom-right
00:15:21.09	panel, but there's... they don't form granulomas.
00:15:25.13	Now, this seemed opposite of what you'd expect, because if... if granulomas are good for the
00:15:32.09	host, as what everyone in TB... in the field of TB thought... people have thought that
00:15:38.12	the granuloma is a critical host protective structure that walls off the bacteria and,
00:15:46.00	while it's not always successful in eradicating the bacteria, it sure as heck tries to do
00:15:51.14	so, and is... is... is pretty good at it.
00:15:54.12	Now, if that's the case, then we should see more granuloma formation with that mutant,
00:16:00.03	but we saw less.
00:16:02.05	So, Hannah took a close look at this and she was able to observe fish as the granuloma
00:16:08.24	formed by serial imaging, and what she showed was that, when the granuloma formed, the number
00:16:16.19	of infected macrophages went up dramatically, as did the number of bacteria.
00:16:21.19	So, the granuloma was actually promoting growth rather than restricting growth.
00:16:26.18	So, why might this be?
00:16:28.15	Because here we are saying that a... the... you know, this... this immunological structure
00:16:34.07	that really should be killing the bacteria is actually promoting growth.
00:16:40.24	And the answer to this came of... both from Hannah's work and from a new graduate student
00:16:49.20	who joined, an MD/PhD student, Muse Davis, and what we found was happening was that,
00:16:57.02	when there's an infected macrophage, when new macrophages come to it, for some reason,
00:17:04.13	if they have that ESX1 locus, the bacteria are spreading quickly from macrophage to macrophage.
00:17:12.16	You can see, within 48 hours, you've gone from one infected macrophage in that top-left
00:17:17.12	panel to many infected macrophages, whereas if you didn't have that locus, if the bacterium
00:17:23.24	didn't have that locus, then that one mac... macrophage just remains one great big macrophage.
00:17:29.09	And the bacteria are just growing in it, but obviously they're not doing as well as if
00:17:33.09	they can spread to new macrophages.
00:17:37.20	And so it turned out that the bacterium is
00:17:40.07	using this locus to spread from one macrophage to another.
00:17:44.16	Now, how does this happen?
00:17:46.13	What... what Muse found was that, if the initial macrophage was infected with bacteria that
00:17:54.03	contained this locus, then somehow that macrophage was able to exert a rapidly chemotactic effect
00:18:03.18	on... on macrophages around it, or... or even far away, so that they came.
00:18:10.18	And you can see that the macrophages in the top panel have these protrusions that reflect
00:18:16.15	that they're highly chemotactic and are responding to a chemotactic gradient, and our racing
00:18:22.13	into this structure.
00:18:24.07	In contrast, if that initial macrophage didn't have this locus, then the incoming macrophages
00:18:31.03	are coming in very, very slowly, and they clearly are not experiencing a chemotactic
00:18:36.15	gradient; they don't have that big... great big protrusion.
00:18:40.18	And, even once they get into the granuloma, they behave very differently.
00:18:45.06	The wild-type macrophages move far and wide within the granuloma, and rapidly, whereas
00:18:50.13	the mutant macrophages, the few that do come, are just sort of sitting there
00:18:54.23	like bumps on a log.
00:18:58.20	And this is the kind of movie that gave us that insight that I just talked to you about,
00:19:04.10	that macrophages are used... exploited by the bacteria to spread from cell to cell.
00:19:11.01	So, what happens is that, when a given infected macrophage packs up with... packs up with
00:19:17.08	bacteria, because the bacteria can grow within it, it undergoes an apoptotic death, where
00:19:24.10	it's dead but it's preserved its membranes.
00:19:27.19	And now what happens is this dead cell is recognized by the incoming macrophages, that
00:19:34.22	come and eat it.
00:19:36.05	And I'm going to show you an example, here, of where one... one dead macrophage with the
00:19:42.07	white arrow is going to be engulfed by an incoming macrophage.
00:19:46.19	So, watch this happen.
00:19:48.02	It's... it... you're going to watch it, it's going to come from below, it's kind of like
00:19:52.04	that movie Jaws, where the... you know, the shark comes from below.
00:19:56.06	And, look at it, it's eating it bite by bite.
00:19:59.22	And this is why macrophages might be called what they are -- macrophage for "big eater".
00:20:05.12	And now you've got... this macrophage has been eaten by a new macrophage.
00:20:12.12	But you're going to say, wait a minute, why would this spread the infection?
00:20:15.18	You've gone from one macrophage to another macrophage, so all you've done is conserved
00:20:19.12	the bacteria.
00:20:21.04	But it turns out, when Muse looked closely, that, on average, a given macrophage, given
00:20:26.14	infected, dead macrophage, was eaten, on average, by 2.3 macrophages every 24 hours.
00:20:34.03	And so you can imagine... you can see how the bug is using the macrophage to expand
00:20:39.24	its numbers.
00:20:42.06	And not only that, but we showed that the bacterium also induces the death of the macrophages.
00:20:47.21	Here's a TUNEL stain on the left and this... this death... there are probably many bacterial
00:20:53.03	determinants that do this, but one of them is that self... same locus, ESX1, that also
00:20:58.11	induces the macrophages to come.
00:21:00.06	So, it's got a two-pronged effect.
00:21:02.05	It's inducing death of the infected macrophage and, separately, it's recruiting new macrophages
00:21:07.18	to come to it so that they can engulf the dead macrophage, and... and produce infection.
00:21:15.22	So, to summarize this, let's take a look at what happens in the mutant first.
00:21:23.19	Because, in the mutant, the granuloma might actually be functioning as a host-protective
00:21:29.14	structure, as it might be meant to be.
00:21:34.06	It's you've... you've got an infected macrophage, it dies at a slow rate, macro... new macrophages
00:21:42.23	are slowly recruited at a... at a respectable pace.
00:21:46.10	And, now, they can eat the new macrophage one by one on one, and there's some time for
00:21:53.07	the cells to also kill the dying cell, to also kill the bacteria before they're eaten,
00:21:59.10	so you could imagine there's an attrition of bacterial infection.
00:22:02.19	This might be why the BCG vaccine strain is attenuated.
00:22:06.11	But, paradoxically, instead of sort of thwarting these host-protective processes, as you might
00:22:14.19	imagine a pathogenic bacterium might do, the... the pathogen actually accelerates these processes,
00:22:21.05	so it converts them from being a host-protective to a host-detrimental process.
00:22:26.04	So, simply by speeding up the rate of cell death, and speeding up the recruitment of
00:22:30.19	new macrophages, it's... it's just using the macrophage niche to spread in from cell to
00:22:37.12	cell, and therefore expand itself in the granuloma.
00:22:40.20	And this is quite a nifty thing to do, I think.
00:22:45.04	So... okay.
00:22:46.13	But the question now is, how do... how do... how does this ESX1 locus induce the recruitment
00:22:53.04	of new macrophages?
00:22:54.21	And so, for this part, Hannah was joined by Tamara Pozos, who was a pediatric infectious
00:23:00.13	diseases fellow who joined the lab, and together they did a microarray where they looked at
00:23:05.10	fish that were infected with wild-type or mutant bacteria to identify host genes that
00:23:11.03	might be different between the two.
00:23:13.00	And the gene that stuck out was matrix metalloproteinase 9.
00:23:17.18	And this is an extracellular... an... an enzyme of that... that models...
00:23:23.06	remodels the extracellular matrix.
00:23:25.22	And they were even able to show, not only by transcriptional analyses but also by doing
00:23:31.06	a gelatinase assay on the fish for the actual activity of this enzyme, that MMP9 was induced
00:23:39.20	upon wild-type infection but not upon mutant infection.
00:23:44.16	Now, that's fine.
00:23:47.03	But if MMP9 is... induction of MMP9 is responsible for the ESX-mediated acceleration of macrophage
00:23:57.13	recruitment, then, if you make a MMP9 mutant, that mutant should be attenuated even with
00:24:03.05	wild-type infection.
00:24:04.08	And, sure enough, when they looked they saw that the MMP9 mutant, which is shown on the
00:24:09.17	bottom, there, that fish, was attenuated for infection, and it had very few granulomas.
00:24:17.15	So, it was behaving just like the bacterial mutant and therefore it was the partner.
00:24:24.09	So, so this was nice and then... but then we started to look into what MMP9 does and
00:24:30.03	then it turns out that MMP9 is involved in the pathogenesis of arthritis, and cancers,
00:24:37.14	and other inflammatory conditions, and often in those cases the mac... it's the mac...
00:24:43.07	it's a macrophage that is the bad actor, that is making MMP9 and... and causing trouble
00:24:49.06	in these lesions.
00:24:50.06	So, of course, we thought, well, okay a macrophage gets infected with the bacteria it now induces
00:24:56.17	MMP9 in the macrophage, and now that... that is secreted and calls in new macrophages.
00:25:03.11	But when we did in situ analyses there... to look at where the MMP9 was being made,
00:25:10.13	here is a granuloma, and the MMP9 is labeled green and the macrophage... macrophages are
00:25:17.14	labeled red, and what you can see is that the MMP9 is not in the... in the macrophages
00:25:22.15	of the granuloma.
00:25:23.17	But, rather, it's in the epithelial cells surrounding the granuloma.
00:25:29.13	And so what... what seems to be happening is that an infected macrophage secretes something
00:25:37.18	from that secretion system that goes and talks to the epithelial cell
00:25:43.02	and induces it to make MMP9.
00:25:45.14	And the MMP9 now calls in new macrophages, and this... this... this strategy was called
00:25:52.09	"subversion from the sidelines" by my friend and colleague, Bill Bishai at Hopkins, and
00:25:57.20	I rather like how he put it.
00:26:00.11	So, why might that be?
00:26:03.05	You could imagine that the bacterium wants to tamp down immunity in the infect... in
00:26:10.05	the actual... in the macrophage itself, because it has to survive in there.
00:26:13.21	So, it's doing that and, meanwhile, it's inducing an inflammatory program in a neighboring cell,
00:26:19.24	so that it can bring more macrophages, infect, and then subvert them.
00:26:25.03	Okay.
00:26:26.04	So, but... but... so this is so... so this is how the bacterium uses the innate immune
00:26:33.23	phase of the granuloma to promote its... its growth and expansion.
00:26:40.14	Of course, then the bac... the granuloma matures and other things happen and, as I told you,
00:26:47.01	one of the things that happens is epithelioid transformation, these tight interdigitated
00:26:52.09	projections, that too has been known for, oh, a hundred years or so, and that very reasonably
00:27:00.01	was thought to be a host-protective mechanism that would sort of wall off the bacteria and...
00:27:09.08	and perhaps be... somehow help the host, despite all these strategies of the bacterium.
00:27:14.06	Well, very recently, work from David Tobin's lab, also done in the zebrafish, that this
00:27:20.11	too turns out not to be the case.
00:27:24.10	Mark Cronin and David Tobin have shown that epithelioid transformation of the macrophage
00:27:31.09	is also something that the bacterium is benefiting from.
00:27:34.16	If they inhibit it, then they can... they get less infection than if it's there.
00:27:43.00	And, of course, they're working out the details of how this might be, but what it's telling
00:27:47.02	you is that practically every step that we might predict will help the host can be taken
00:27:52.21	advantage of by the bacterium.
00:27:56.04	So, in my first lecture, I told you that most people actually clear infection and they do
00:28:02.11	so after the adaptive phase of the... of the granuloma has kicked in.
00:28:06.22	So, it's very clear that... that at some point the granuloma can fight back and can... can
00:28:14.21	eradicate or at least suppress infection.
00:28:17.14	And many, many people who specialize in the areas of adaptive immunity and TB are... are
00:28:24.17	working on this.
00:28:26.14	But I want to close by saying that, while the adaptive immunologists may not know as
00:28:32.11	much about this as they want to, and they're working hard to figure it out, the bacterium
00:28:37.01	seems to know quite a little... quite a bit about this... these immune mechanisms, because
00:28:42.11	what these people so... who worked on it so far can tell you is that it tries really hard
00:28:48.08	to delay and inhibit adaptive immunity, so that the adaptive immune elements that come
00:28:54.18	into the granuloma do so late.
00:28:58.10	And this gives time for the mechanisms that I've just been telling you about to help bacteria
00:29:04.20	expand in the innate context.
00:29:07.20	I'll close by thanking the many people whose research I've described to you -- they're
00:29:14.04	both students and from my lab, as well as colleagues and collaborators from outside
00:29:19.19	my lab, and, indeed, from across the world.
00:29:23.08	Thank you.